Inbred miniature swine provide a unique preclinical model for the study of transplantation immunity and tolerance. We have previously demonstrated that robust tolerance to MHC class l-mismatched renal allografts can be achieved following a short course of calcineurin inhibitors in this model. Since, unlike central tolerance, the induction of tolerance in this system does not involve T cell ablation, and since T cells are long-lived, the central hypothesis of this proposal is that this form of tolerance involves a continuing mechanism for down-regulation of T cell reactivity. During the last project period, our inbreeding program has produced a subline with >94% coefficient of inbreeding, permitting investigation of the mechanism of tolerance by adoptive transfer for the first time in a large animal model. Our data using these animals indicate that tolerance to class I mismatched renal allografts involves regulatory T cells (T-reg) that can be isolated from the long-term tolerated kidney and can also be mobilized in the periphery following DST. We have also observed that tolerance persists for at least 3-months after removal of the graft, and that during this period, immunization by donor class I peptides, but not by rejection of donor skin grafts, abrogates the tolerant state. Finally, we have found that the treatment of tolerant animals with DST and leukapheresis, required for successful adoptive transfer of their tolerance, also leads to loss of the tolerant state. Collectively, these data suggest that tolerance relies on a balance between alloreactivity and regulation, which is maintained via definable cellular interactions between the graft and the recipient's immune system. To test these hypotheses, we will 1) Determine the nature of the cell populations responsible for transfer of tolerance by adoptive transfer;2) Study the mechanism by which the pathway of antigen presentation determines maintenance vs. loss of tolerance;and 3) Examine the balance between alloreactivity and regulation that determines the fate of a second transplant into a tolerant recipient. It is hoped that an understanding of the mechanisms by which allograft tolerance is induced and maintained in this large-animal model will permit development of appropriate clinical protocols for induction of specific tolerance to organ allografts.